Compositions and methods for treating viral infections

ABSTRACT

The present invention provides compositions methods for treating susceptible viral infections, especially hepatitis C viral (HCV) infections as well as co infections of HCV with other viruses such as HBV and/or HIV. In one embodiment, the present invention provides compositions having the formula (I) and their use in treating viral infections: 
     
       
         
         
             
             
         
       
         
         
           
             or a pharmaceutically acceptable salt, ester, stereoisomer, tautomers, solvate, prodrug, or combination thereof.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/800,931, filed Mar. 13, 2013, which is a continuation of U.S. Pat.No. 8,404,651, filed Oct. 4, 2010, which is a continuation ofInternational Application No. PCT/US2009/039424, filed Apr. 3, 2009,which claims the benefit of U.S. Provisional Application No. 61/072,794,filed Apr. 3, 2008; U.S. Provisional Application No. 61/072,799, filedApr. 3, 2008; and U.S. Provisional Application No. 61/074,421, filedJun. 20, 2008. The entire teachings of the above applications areincorporated herein by reference.

BACKGROUND OF THE INVENTION

Over 170 million people worldwide are infected by Hepatitis C virus. Thecurrent therapies have dose-limiting toxicity and there existssignificant unmet medical need. Several compounds are under developmentthat target HCV polymerase, HCV protease, and HCV NS5A. However, theviral replication cycle is significantly error-prone resulting inemergence of resistant mutants particularly under the selective pressureof antiviral therapy. This presents significant challenges to developingantiviral treatment regimes.

To date most anti-HCV agents that have been developed are HCV polymeraseinhibitors. An NS3 protease inhibitor BILN-2061 was the first compoundtested in humans that produced significant viral load reduction inpatients. A nucleoside analog NM 283 showed antiviral effect inHCV-infected patients. However significant drug resistance and toxicityhave been already noted with a few compounds in the clinic. Severalantiviral compounds are under clinical development. For example,non-nucleoside benzothiadiazines, acyl pyrrolidines, benzofurans,phenylalanines, substituted thiophene, dihydropyranones, pyranoindoles,benzimidazoles and indole have been found to be inhibitors of NS5Bpolymerase domain. However, in vitro replicon assays reveal significantcross resistance with different drugs.

Because resistance development to antiviral drugs is virtually certain,one way to combat it has been combination treatments including drugsthat may not promote cross-resistant mutants. Usually drugs that effectdifferent viral enzymes do not show cross resistance and can be used incombinations successfully. Thus, a combination of different drugs withdifferent mechanisms of action, which are not cross-resistant, will bethe key to successful antiviral therapy.

Shorter chain oligonucleotides (less than 8-mers) with lesser number ofcharges and smaller molecular weight compared to 20-mer oligonucleotidesrepresent a promising class of novel molecules with potentialtherapeutic properties as antivirals. Indeed, recent reports suggestthat mono-, di-, tri-, and short chain oligonucleotides possesssignificant biological activity that can be exploited for varioustherapeutic applications. However, improved short oligonucleotideshaving improved properties for oral, transdermal or other non-invasivemodes of delivery to the patient are still needed for use as stand alonetherapeutics or in combination therapies.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for treatingsusceptible viral infections, especially hepatitis C viral (HCV)infections as well as co-infections of HCV with other viruses such asHBV and/or HIV. In one embodiment, the present invention providescompositions having the formula (I) and their use in treating viralinfections:

or a pharmaceutically acceptable salt, ester, stereoisomer, tautomers,solvate, prodrug, or combination thereof, wherein:

N₁ and N₂ are independently selected from naturally occurringnucleosides or modified nucleosides;

W, X, Y and Z are each independently selected from O, S and NR₁ whereinR₁ is independently selected from hydrogen, substituted or unsubstitutedaliphatic and substituted or unsubstituted aromatic;

R₂, R₃, R₄ and R₅ are each independently selected from hydrogen,substituted or unsubstituted aliphatic group and substituted orunsubstituted aromatic group;

n is 0, 1, 2, 3, 4 or 5;

A is absent, or substituted or unsubstituted aromatic group;

J is absent, CR₆R₇, O, S or NR₁ wherein R₆ and R₇ are each independentlyselected from hydrogen, substituted or unsubstituted aliphatic group andsubstituted or unsubstituted aromatic group and R₁ is as defined above;

M is absent, CR₈R₉, O, S or NR₁ wherein R₈ and R₉ are each independentlyselected from hydrogen, substituted or unsubstituted aliphatic group andsubstituted or unsubstituted aromatic group and R₁ is as defined above;

V is substituted or unsubstituted aliphatic group or substituted orunsubstituted aromatic group;

Q is absent, or a modified or unmodified nucleotide; and

m is 1, 2, 3 or 4.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides short nucleotide compositions and methodsfor treating susceptible viral infections, especially hepatitis C viralinfections. The term “short nucleotide(s)” refers to a mononucleotide,dinucleotide or polynucleotide formed from 1 to about 6 linkednucleoside units. The invention also encompasses mononucleosidecompounds. The term “susceptible viral infections” as used herein meansviral infections caused by a wide range of RNA and DNA viruses,including, but not limited to, the families of viruses such asflaviviruses-including the genus flavivirus, pestivirus of which Kunjinvirus is a member, and hepavirus of which hepatitis C virus is a member,and arbovirus of which the West Nile virus is a member,orthomyxoviruses, paramyxoviruses, arenaviruses, bunyaviruses, herpesviruses, adenoviruses, poxviruses, and retroviruses. Typical suitable“susceptible viral infections” include influenza A and B viralinfections; parainfluenza viral infections, respiratory syncytial virus(“RSV”) infections such as RSV bronchiolitis and RSV pneumoniaespecially such RSV infections in children and infants as well as RSVpneumonia in patients with preexisting cardiopulmonary disease, measlesviral infections, Lassa fever viral infections, Korean Haemorrhagicfever infections, hepatitis B viral (HBV) infections, CrimeanCongo-Haemorrhagic and HCV infections and HIV-1 infections, encephalitisinfections such as caused by West Nile virus or Kunjin virus or the St.Louis encephalitis infections, as well as viral infections found inimmunocompromised patients. Other susceptible viral infections aredisclosed in U.S. Pat. No. 4,211,771 at column 2, line 21 to column 3line 37; doses and dose regimens and formulations are disclosed atcolumn 3, line 4 to column 9, line 5. In one embodiment, the viralinfection is not solely an HBV infection, however, the viral infectionmay be an HBV co-infection with another virus such as HCV.

The compositions of the invention suitable for treating susceptibleviral infections and particularly HCV infections as well as coinfections of HCV with other viruses such as HBV and/or HIV, arerepresented by compounds of formulas I-IV. It should be noted that insome of the following formulas, a nucleoside unit is represented by theinternationally accepted convention of line drawing. In the examplebelow a 2′-substituted ribonucleoside is represented in both theconventional structure and the corresponding line drawing format:

The sugar units attached to B₁ and B₂ that give rise to α or β N- orC-nucleoside includes, but is not limited to, furanose,deoxyribofuranose, ribose, and arabinose.

In a first embodiment, the compounds of the present invention arecompounds represented by formula I illustrated above, or racemates,enantiomers, diastereomers, geometric isomers, tautomers thereof.

A preferred subgenera of formula I are compounds represented by formula(II) as illustrated below, or racemates, enantiomers, diastereomers,geometric isomers, tautomers thereof.

Wherein V, M, J, A, R₂, R₃, R₄, R₅, N₁, N₂, Q, m and n are as previouslydefined in formula I.

Representative compounds according to the invention are those selectedfrom the group consisting of: Compounds (1)-(8) of the formula A1:

Wherein V, M, R₁₀ and R₁₁ are delineated for each example in Table 1.

TABLE 1 Compound No. V M R₁₀ R₁₁ 1

absent H H 2

O H H 3

absent H H 4

O H H 5

O C(O)Ph H 6

O H C(O)Ph 7

absent H H 8

O H H

Representative compounds according to the invention are those selectedfrom the group consisting of:

Compounds (9)-(16) of the formula B1:

wherein V, M, R₁₀ and R₁₁ are delineated for each example in Table 2.

TABLE 2 Compound No. V M R₁₀ R₁₁  9

absent H H 10

O H H 11

absent H H 12

O H H 13

O C(O)Ph H 14

O H C(O)Ph 15

absent H H 16

O H HExamples of compounds of formula I include but are not limited to:

Other examples of compounds of Formula I include but are not limited to:

wherein B₁ and B₂ are naturally occurring nucleobases or modified bases.

In another embodiment, the present invention provides compounds havingthe formula (III) and their use in treating viral infections:

or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer,solvate, prodrug, or combination thereof, wherein:M′ and M″ are each independently selected from CH₂, NH, NR″, O, and S;wherein R″ is substituted or unsubstituted aliphatic group orsubstituted or unsubstituted aromatic group;X′ is O, NH, NR″, or S; wherein R″ is as previously defined;Y′ is OR₁₂, NHR₁₂, and SR₁₂; where each R₁₂ is independently selectedfrom H, substituted or unsubstituted aliphatic group or substituted orunsubstituted aromatic group;Z′ and Z″ are each independently O, NR₁₃, and S; where R₁₃ is H,substituted or unsubstituted aliphatic group or substituted orunsubstituted aromatic group;R and R′ are each independently H, OH, O-alkyl, O-aryl, O-heteroaryl,O-aralkyl, O-alkyl heteroaryl, —NH₂, —NHR₁₄, —NR₁₅NR₁₆, substituted orunsubstituted alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted aryl, substituted or unsubstituted aralkyl,or substituted or unsubstituted heterocyclic, wherein R₁₄, R₁₅ and R₁₆are each independently selected from H, substituted or unsubstitutedaliphatic group or substituted or unsubstituted aromatic group;B₁ and B₂ are each independently selected from absent, H, naturallyoccurring nucleobases and modified bases; andQ′ is absent or

wherein X′ and Y′ are as previously defined and A is OH, O-alkyl,O-aryl, O-heteroaryl, O-aralkyl, or O-alkyl heteroaryl.

In a preferred embodiment, at least one of B₁ and B₂ are independentlythe following formula:

Specific examples of compounds of Formula III include but are notlimited to:

wherein in the above formulas R is a substituted or unsubstitutedaliphatic group having a backbone of up to ten atoms and preferably, Ris a long chain fatty acid.

Additional compounds of formula III include but are not limited to thefollowing:

wherein in those above formulas B₁ and B₂ are independently naturallyoccurring nucleobases or modified bases.

In another embodiment, the present invention provides compounds havingthe formula (IV) and their use in treating viral infections:

M′ is selected from CH₂, NH, NR″, O, and S; wherein R″ is substituted orunsubstituted aliphatic group or substituted or unsubstituted aromaticgroup;X is O, NH, NR″, or S where R″ is previously defined;Z′ is H, OH, OR″, OR₁₇, COOH, COOR″, NH₂, NHR″, NHR₁₇ where R″ ispreviously defined and R₁₇ is aroyl (CO-Ph), sulfonyl (SO₂—R), ureidyl(CO—NH—R), thioureidyl (CS—NH—R) wherein R is selected from hydrogen,substituted or unsubstituted aliphatic group and substituted orunsubstituted aromatic group, however when M′ is O, Z′ is not OH;R′ is H, OH, O-alkyl, O-aryl, O-heteroaryl, O-aralkyl, O-alkylheteroaryl, O-aroyl, —NH₂, —NHR₁, —NR₁NR₂ alkyl, substituted alkyl,cycloalkyl, aryl, substituted aryl, aralkyl, or heterocylic wherein R₁and R₂ are each independently selected from hydrogen, substituted orunsubstituted aliphatic group and substituted or unsubstituted aromaticgroup, however when M′ is O, R′ is not OH;B₁ is H, a naturally occurring nucleobase or a modified base.

In a preferred embodiment, R, R₁ and R₂ of formula IV are substituted orunsubstituted alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted aryl, substituted or unsubstituted aralkyl,or substituted or unsubstituted heterocyclic.

Non limiting examples of compounds of formula (IV) include:

Listed below are definitions of various terms used to describe thisinvention. These definitions apply to the terms as they are usedthroughout this specification and claims, unless otherwise limited inspecific instances, either individually or as part of a larger group.

An “aliphatic group” is non-aromatic moiety that may contain anycombination of carbon atoms, hydrogen atoms, halogen atoms, oxygen,nitrogen, sulfur or other atoms, and optionally contain one or moreunits of unsaturation, e.g., double and/or triple bonds. An aliphaticgroup may be straight chained, branched or cyclic and preferablycontains between about 1 and about 24 carbon atoms, more typicallybetween about 1 and about 12 carbon atoms. In addition to aliphatichydrocarbon groups, aliphatic groups include, for example,polyalkoxyalkyls, such as polyalkylene glycols, polyamines, andpolyimines, for example. It is understood that any alkyl, alkenyl,alkynyl and cycloalkyl moiety described herein can also be an aliphaticgroup, an alicyclic group or a heterocyclic group. Such aliphatic groupsmay be further substituted.

The term “aryl,” as used herein, refers to a mono- or polycycliccarbocyclic ring system including, but not limited to, phenyl, naphthyl,tetrahydronaphthyl, indanyl, idenyl. The term aryl includes, but is notlimited to, bicyclic aryls or bicyclic heteroaryls having a ring systemconsisting of two rings wherein at least one ring is aromatic. The termaryl includes, but is not limited to, tricyclic aryls or tricyclicheteroaryls having a ring system consisting of three rings wherein atleast one ring is aromatic.

The term “heteroaryl,” as used herein, refers to a mono- or polycyclicaromatic radical having one or more ring atom selected from S, O and N;and the remaining ring atoms are carbon, wherein any N or S containedwithin the ring may be optionally oxidized. Heteroaryl includes, but isnot limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl,imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl,thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl,benzooxazolyl, quinoxalinyl.

In accordance with the invention, any of the aryls, substituted aryls,heteroaryls and substituted heteroaryls described herein, can also beany aromatic group. Therefore, the term “aromatic group” as used hereinincludes all such aryls, substituted aryls, heteroaryls and substitutedheteroaryls. Aromatic groups can be substituted or unsubstituted.

The term “alkyl,” as used herein, refers to saturated, straight- orbranched-chain hydrocarbon radicals containing one or more carbon atomsand preferably contain 1-24 carbon atoms. Examples of alkyl radicalsinclude, but are not limited to, methyl, ethyl, propyl, isopropyl,n-butyl, tert-butyl, neopentyl, n-hexyl, heptyl and octyl radicals,decyl, dodecyl radicals.

The term “alkenyl,” as used herein, refer to straight- or branched-chainhydrocarbon radicals containing at least two and preferably from two toeight carbon atoms having at least one carbon-carbon double bond by theremoval of a single hydrogen atom. Alkenyl groups include, but are notlimited to, for example, ethenyl, propenyl, butenyl,1-methyl-2-buten-1-yl, heptenyl, octenyl, and the like.

The term “alkynyl,” as used herein, refer to straight- or branched-chainhydrocarbon radicals containing at least two carbon atoms and preferablyfrom two to eight carbon atoms having at least one carbon-carbon triplebond by the removal of a single hydrogen atom. Representative alkynylgroups include, but are not limited to, for example, ethynyl,1-propynyl, 1-butynyl, heptynyl, octynyl, and the like.

The term “cycloalkyl” as used herein, refers to a monocyclic orpolycyclic saturated carbocyclic ring compound. Examples of cycloalkylinclude, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl and cyclooctyl; bicyclo[2.2.1]heptyl, andbicyclo[2.2.2]octyl.

The term “cycloalkenyl” as used herein, refers to monocyclic orpolycyclic carbocyclic ring compound having at least one carbon-carbondouble bond. Examples of cycloalkenyl include, but not limited to,cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl,cyclooctenyl, and the like.

It is understood that any alkyl, alkenyl, alkynyl and cycloalkyl moietydescribed herein can also be an aliphatic group as defined above, analicyclic group or a heterocyclic group.

The term “alicyclic,” as used herein, denotes a monovalent group derivedfrom a monocyclic or bicyclic saturated carbocyclic ring compound by theremoval of a single hydrogen atom. Examples include, but not limited to,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[2.2.1]heptyl,and bicyclo[2.2.2]octyl. Such alicyclic groups may be furthersubstituted.

The terms “heterocyclic” or “heterocycloalkyl” can be usedinterchangeably and referred to a non-aromatic ring or a bi- ortri-cyclic group fused system, where (i) each ring system contains atleast one heteroatom independently selected from oxygen, sulfur andnitrogen, (ii) each ring system can be saturated or unsaturated (iii)the nitrogen and sulfur heteroatoms may optionally be oxidized, (iv) thenitrogen heteroatom may optionally be quaternized, (v) any of the aboverings may be fused to an aromatic ring, and (vi) the remaining ringatoms are carbon atoms which may be optionally oxo-substituted.Representative heterocyclic groups include, but are not limited to,1,3-dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl,imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl,morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl,pyridazinonyl, and tetrahydrofuryl. Such heterocyclic groups may befurther substituted.

The term “substituted” refers to substitution by independent replacementof one, two, or three or more of the hydrogen atoms on a moiety such asan aromatic group or an aliphatic group with substituents including, butnot limited to, —F, —Cl, —Br, —I, —OH, protected hydroxy, —NO₂, —CN,—NH₂, protected amino, oxo, thioxo, steroidal, —NH—C₁-C₁₂-alkyl,—NH—C₂-C₈-alkenyl, —NH—C₂-C₈-alkynyl, —NH—C₃-C₁₂-cycloalkyl, —NH-aryl,—NH-heteroaryl, —NH-heterocycloalkyl, -dialkylamino, -diarylamino,-diheteroarylamino, —O—C₁-C₁₂-alkyl, —O—C₂-C₈-alkenyl, —O—C₂-C₈-alkynyl,—O—C₃-C₁₂-cycloalkyl, —O-aryl, —O-heteroaryl, —O— heterocycloalkyl,—C(O)—C₁-C₁₂-alkyl, —C(O)—C₂-C₈-alkenyl, —C(O)—C₂-C₈-alkynyl,—C(O)—C₃-C₁₂-cycloalkyl, —C(O)-aryl, —C(O)-heteroaryl,—C(O)-heterocycloalkyl, —CONH₂, —CONH—C₁-C₁₂-alkyl, —CONH—C₂-C₈-alkenyl,—CONH—C₂-C₈-alkynyl, —CONH—C₃-C₁₂-cycloalkyl, —CONH-aryl,—CONH-heteroaryl, —CONH-heterocycloalkyl, —OCO₂—C₁-C₁₂-alkyl,—OCO₂—C₂-C₈-alkenyl, —OCO₂—C₂-C₈-alkynyl, —OCO₂—C₃-C₁₂-cycloalkyl,—OCO₂-aryl, —OCO₂-heteroaryl, —OCO₂-heterocycloalkyl, —OCONH₂,—OCONH—C₁-C₁₂-alkyl, —OCONH—C₂-C₈-alkenyl, —OCONH—C₂-C₈-alkynyl,—OCONH—C₃-C₁₂-cycloalkyl, —OCONH-aryl, —OCONH-heteroaryl,—OCONH-heterocycloalkyl, —NHC(O)—C₁-C₁₂-alkyl, —NHC(O)—C₂-C₈-alkenyl,—NHC(O)—C₂-C₈-alkynyl, —NHC(O)—C₃-C₁₂-cycloalkyl, —NHC(O)-aryl,—NHC(O)-heteroaryl, —NHC(O)— heterocycloalkyl, —NHCO₂—C₁-C₁₂-alkyl,—NHCO₂—C₂-C₈-alkenyl, —NHCO₂—C₂-C₈-alkynyl, —NHCO₂—C₃-C₁₂-cycloalkyl,—NHCO₂-aryl, —NHCO₂-heteroaryl, —NHCO₂-heterocycloalkyl, —NHC(O)NH₂,—NHC(O)NH—C₁-C₁₂-alkyl, —NHC(O)NH—C₂-C₈-alkenyl,—NHC(O)NH—C₂-C₈-alkynyl, —NHC(O)NH—C₃-C₁₂-cycloalkyl, —NHC(O)NH-aryl,—NHC(O)NH-heteroaryl, —NHC(O)NH-heterocycloalkyl, NHC(S)NH₂,—NHC(S)NH—C₁-C₁₂-alkyl, —NHC(S)NH—C₂-C₈-alkenyl,—NHC(S)NH—C₂-C₈-alkynyl, —NHC(S)NH—C₃-C₁₂-cycloalkyl, —NHC(S)NH-aryl,—NHC(S)NH-heteroaryl, —NHC(S)NH-heterocycloalkyl, —NHC(NH)NH₂,—NHC(NH)NH—C₁-C₁₂-alkyl, —NHC(NH)NH—C₂-C₈-alkenyl,—NHC(NH)NH—C₂-C₈-alkynyl, —NHC(NH)NH—C₃-C₁₂-cycloalkyl, —NHC(NH)NH-aryl,—NHC(NH)NH-heteroaryl, —NHC(NH)NH— heterocycloalkyl,—NHC(NH)—C₁-C₁₂-alkyl, —NHC(NH)—C₂-C₈-alkenyl, —NHC(NH)—C₂-C₈-alkynyl,—NHC(NH)—C₃-C₁₂-cycloalkyl, —NHC(NH)-aryl, —NHC(NH)-heteroaryl,—NHC(NH)-heterocycloalkyl, —C(NH)NH—C₁-C₁₂-alkyl,—C(NH)NH—C₂-C₈-alkenyl, —C(NH)NH—C₂-C₈-alkynyl,—C(NH)NH—C₃-C₁₂-cycloalkyl, —C(NH)NH-aryl, —C(NH)NH-heteroaryl,—C(NH)NH-heterocycloalkyl, —S(O)—C₁-C₁₂-alkyl, —S(O)—C₂-C₈-alkenyl,—S(O)—C₂-C₈-alkynyl, —S(O)—C₃-C₁₂-cycloalkyl, —S(O)-aryl,—S(O)-heteroaryl, —S(O)-heterocycloalkyl-SO₂NH₂, —SO₂NH—C₁-C₁₂-alkyl,—SO₂NH—C₂-C₈-alkenyl, —SO₂NH—C₂-C₈-alkynyl, —SO₂NH—C₃-C₁₂-cycloalkyl,—SO₂NH-aryl, —SO₂NH-heteroaryl, —SO₂NH-heterocycloalkyl,—NHSO₂—C₁-C₁₂-alkyl, —NHSO₂—C₂-C₈-alkenyl, —NHSO₂—C₂-C₈-alkynyl,—NHSO₂—C₃-C₁₂-cycloalkyl, —NHSO₂-aryl, —NHSO₂-heteroaryl,—NHSO₂-heterocycloalkyl, —CH₂NH₂, —CH₂SO₂CH₃, -aryl, -arylalkyl,-heteroaryl, -heteroarylalkyl, -heterocycloalkyl, —C₃-C₁₂-cycloalkyl,polyalkoxyalkyl, polyalkoxy, -methoxymethoxy, -methoxyethoxy, —SH,—S—C₁-C₁₂-alkyl, —S—C₂-C₈-alkenyl, —S—C₂-C₈-alkynyl,—S—C₃-C₁₂-cycloalkyl, —S-aryl, —S-heteroaryl, —S-heterocycloalkyl, ormethylthiomethyl. It is understood that the aryls, heteroaryls, alkyls,steroidals and the like can be further substituted.

The term “halogen,” as used herein, refers to an atom selected fromfluorine, chlorine, bromine and iodine.

The term “steroidal”, as used herein, refers to any of numerousnaturally occurring or synthetic fat-soluble organic compounds having asa basis 17 carbon atoms arranged in four rings and including the sterolsand bile acids, adrenal and sex hormones, certain natural drugs such asdigitalis compounds, and the precursors of certain vitamins. Examples ofsteroidal structure include, but are not limited to, cholesterol,cholestanol, 3α-cyclo-5-α-cholestan-6-β-ol, cholic acid, cholesterylformiate, cholestanyl formiate.

For purposes of the invention, the term “short nucleotide(s)” refers toa mono, di or polynucleoside formed from 1 to about 6 linked nucleosideunits. Such short nucleotides can be obtained from existing nucleic acidsources, including genomic or cDNA, but are preferably produced bysynthetic methods. The nucleoside residues can be coupled to each otherby any of the numerous known internucleoside linkages. Suchinternucleoside linkages may be modified or unmodified and include,without limitation, phosphodiester, phosphorothioate,phosphorodithioate, alkylphosphonate, alkylphosphonothioate,phosphotriester, phosphoramidate, siloxane, carbonate, carboalkoxy,acetamidate, carbamate, morpholino, borano, thioether, bridgedphosphoramidate, bridged methylene phosphonate, bridgedphosphorothioate, and sulfone internucleoside linkages. The term “shortnucleotide” also encompasses polynucleosides having one or morestereospecific internucleoside linkage (e.g., (R_(P))- or(S_(P))-phosphorothioate, alkylphosphonate, or phosphotriester linkages.The short nucleotides of the invention include any such internucleosidelinkage, whether or not the linkage comprises a phosphate group. Incertain preferred embodiments, these internucleoside linkages may bemodified or unmodified and include without limitation, phosphodiester,phosphorothioate, or phosphorodithioate linkages, or combinationsthereof.

The term “short nucleotide(s)” also encompasses additional substituentsincluding, without limitation, protein groups, lipophilic groups,intercalating agents, diamines, folic acid, cholesterol and adamantane.

The term “short nucleotide(s)” also encompasses any other nucleobasecontaining polymers, including, without limitation, peptide nucleicacids (PNA), peptide nucleic acids with phosphate groups (PHONA), lockednucleic acids (LNA). Examples of PNA and LNA are shown below:

A “nucleotide” refers to a sub-unit of a nucleic acid (whether DNA orRNA or analogue thereof) which includes an internucleotide linkage, asugar group and a heterocyclic base, as well as analogs of suchsub-units. A “nucleoside” references a nucleic acid subunit including asugar group and a heterocyclic base. It will be appreciated that, asused herein, the terms “nucleoside” and “nucleotide” will include thosemoieties which contain not only the naturally occurring internucleotidelinkages (with respect to “nucleotides”) such as phosphodiesterinternucleotide linkage; naturally occurring sugar moieties such as aribose and deoxyribose moieties; and naturally occurring nucleobasessuch as purine and pyrimidine bases, e.g., adenine (A), thymine (T),cytosine (C), guanine (G), or uracil (U), but also modifiedinternucleotide linkages, modified sugar moieties and modified purineand pyrimidine bases or analogs thereof or any combination of modifiedand unmodified internucleotide linkage, sugar moiety and purine andpyrimidine bases. Other examples of modified nucleosides includeacyclonucleosides, which consists of ring-opened versions of the riboseand deoxyribose moieties. Correspondingly, such ring opened nucleosidesmay be used in forming modified nucleotides. Other examples of modifiednucleosides include C-nucleosides such as pseudoisocytidine, andnucleoside mimics including nucleoside isosteres such as peptide nucleicacid monomers.

Modifications of naturally occurring purine and pyrimidine nucleobasesinclude but are not limited to methylated purines or pyrimidines,acylated purines or pyrimidines, and the like, or the addition of aprotecting group such as acetyl, difluoroacetyl, trifluoroacetyl,isobutyryl, benzoyl, or the like. The purine or pyrimidine base may alsobe an analog of the foregoing; suitable analogs will be known to thoseskilled in the art and are described in the pertinent texts andliterature. Common analogs include, but are not limited to,1-methyladenine, 2-methyladenine, N6-methyladenine, N6-isopentyladenine,2-methylthio-N-6-isopentyladenine, N,N-dimethyladenine, 8-bromoadenine,2-thiocytosine, 3-methylcytosine, 5-methylcytosine, 5-ethylcytosine,4-acetylcytosine, 1-methylguanine, 2-methylguanine, 7-methylguanine,2,2-dimethylguanine, 8-bromoguanine, 8-chloroguanine, 8-aminoguanine,8-methylguanine, 8-thioguanine, 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, 5-ethyluracil, 5-propyluracil,5-methoxyuracil, 5-hydroxymethyluracil, 5-(carboxyhydroxymethyl)uracil,5-(methylaminomethyl)uracil, 5-(carboxymethylaminomethyl)-uracil,2-thiouracil, 5-methyl-2-thiouracil, 5-(2-bromovinyl) uracil,uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester,pseudouracil, 1-methylpseudouracil, queosine, inosine, 1-methylinosine,hypoxanthine, xanthine, 2-aminopurine, 6-hydroxyaminopurine,6-thiopurine, 2,6-diaminopurine 5-trifluoromethyl thymine,6-chloro-adenine, 7-deaza-adenine.

It should also be understood that a “modified base” also referred to asa “modified nucleobase”, includes a nitrogen containing compound thatmay or may not be heterocyclic. Such preferred nitrogen containingcompounds include but are not limited to —NHR₁₈ wherein R₁₈ is hydrogen,butyloxycarbonyl (Boc), benzyloxycarbonyl, allyl, substituted orunsubstituted alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted aralkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, or heterocyclic.

The term “modified base” is further intended to include heterocycliccompounds that that are not nucleosidic bases in the most classicalsense but that can serve as nucleosidic bases. Such compounds include“universal bases” as are known in the art. Universal bases may includean aromatic ring moiety, which may or may not contain nitrogen atoms. Insome embodiments, a universal base may be covalently attached to theC-1′ carbon of a pentose sugar of the nucleoside. Examples of universalbases include 3-methyl-propynylcarbostyryl (PIM), 3-methylisocarbostyryl(MICS), and 5-methyl isocarbostyryl moieties. Additional examplesinclude Inosine derivatives, azole carboxamide analogues, nitroazoles,and nitroimidazoles.

Examples of modified nucleotide and nucleoside sugar moieties includebut are not limited to: trehalose, arabinose, 2′-deoxy-2′-substitutedpentose moiety, 2′-O-substituted pentose moiety, lyxose, and xylose, orhexose sugar group. For purposes of the invention, the term“2′-substituted” of any of the named sugar groups such as “2′substitutedribonucleoside” or “2′-substituted arabinoside” includes ribonucleosidesor arabinonucleoside in which the hydroxyl group at the 2′ position ofthe pentose moiety is substituted to produce a 2′-substituted or2′-O-substituted ribonucleoside or arabinonucleoside. Preferably, suchsubstitution is with a lower alkyl group containing 1-6 saturated orunsaturated carbon atoms, or with an aryl group having 6-10 carbonatoms, wherein such alkyl, or aryl group may be unsubstituted or may besubstituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro,acyl, acyloxy, alkoxy, carboxyl, carboalkoxy, or amino groups. Examplesof 2′-O-substituted ribonucleosides or 2′-O-substituted-arabinosidesinclude, without limitation 2′-O-methylribonucleosides (also indicatedherein as 2′-OMe) or 2′-O-methylarabinosides and2′-O-methoxyethylribonucleosides or 2′-O-methoxyethylarabinosides. Theterm “2′-substituted ribonucleoside” or “2′-substituted arabinoside”also includes ribonucleosides or arabinonucleosides in which the2′-hydroxyl group is replaced with a lower alkyl group containing 1-6saturated or unsaturated carbon atoms, or with an amino or halo group.Examples of such 2′-substituted ribonucleosides or 2′-substitutedarabinosides include, without limitation, 2′-amino, 2′-fluoro, 2′-allyl,and 2′-propargyl ribonucleosides or arabinosides.

Examples of modified internucleotide linkages include but are notlimited to: substituted and unsubstituted phosphorothioate,phosphorodithioate, alkylphosphonate, alkylphosphonothioate,phosphotriester, phosphoramidate, siloxane, carbonate, carboalkoxy,acetamidate, carbamate, morpholino, borano, thioether, bridgedphosphoramidate, bridged methylene phosphonate, bridgedphosphorothioate, and sulfone internucleoside linkages. Substitutions ofmodified and unmodified internucleotide linkages include the followingmoiety:

wherein V, M, J, R₂, R₃, R₄, R₅ and n are all previously defined informula I.

The compounds of the present invention contain one or more asymmetriccenters and can thus occur as racemates and racemic mixtures, singleenantiomers, diastereomeric mixtures and individual diastereomers. Thepresent invention is meant to include short nucleotide compounds havingthe β-D stereochemical configuration for the five-membered furanosering, that is, short nucleotide compounds in which the substituents atC-1 and C-4 of the five-membered furanose ring have the β-stereochemicalconfiguration (“up” orientation which is typically denoted by a boldline in some formulas depicted herein).

ABBREVIATIONS

Abbreviations which may be used in the descriptions of the scheme andthe examples that follow are:

Ac for acetyl;

AcOH for acetic acid;

Boc₂O for di-tert-butyl-dicarbonate;

Boc for t-butoxycarbonyl;

Bpoc for 1-methyl-1-(4-biphenylyl)ethyl carbonyl;

Bz for benzoyl;

Bn for benzyl;

BocNHOH for tert-butyl N-hydroxycarbamate;

t-BuOK for potassium tert-butoxide;

Bu₃SnH for tributyltin hydride;

CDI for carbonyldiimidazole;

CH₂Cl₂ for dichloromethane;

CH₃ for methyl;

CH₃CN for acetonitrile;

DMSO for dimethyl sulfoxide;

EtOAc for ethyl acetate;

EtOH for ethanol;

Et₂O for diethyl ether;

HCl for hydrogen chloride;

MeOH for methanol;

MOM for methoxymethyl;

Ms for mesyl or —SO₂—CH₃;

Ms₂O for methanesulfonic anhydride or mesyl-anhydride;

NaCl for sodium chloride;

NaH for sodium hydride;

NaHCO₃ for sodium bicarbonate or sodium hydrogen carbonate;

Na₂CO₃ sodium carbonate;

NaOH for sodium hydroxide;

Na₂SO₄ for sodium sulfate;

NaHSO₃ for sodium bisulfite or sodium hydrogen sulfite;

Na₂S₂O₃ for sodium thiosulfate;

NH₂NH₂ for hydrazine;

NH₄HCO₃ for ammonium bicarbonate;

NH₄Cl for ammonium chloride;

OH for hydroxyl;

OMe for methoxy;

OEt for ethoxy;

TEA or Et₃N for triethylamine;

TFA trifluoroacetic acid;

THF for tetrahydrofuran;

TPP or PPh₃ for triphenylphosphine;

Ts for tosyl or —SO₂—C₆H₄CH₃;

Ts₂O for tolylsulfonic anhydride or tosyl-anhydride;

TsOH for p-tolylsulfonic acid;

Ph for phenyl;

TBS for tert-butyl dimethylsilyl;

TMS for trimethylsilyl; or

TMSCl for trimethylsilyl chloride.

Also included within the present invention are pharmaceuticalcompositions comprising the short oligonucleotide compounds of theinvention and derivatives thereof of the present invention inassociation with a pharmaceutically acceptable carrier. Another exampleof the invention is a pharmaceutical composition made by combining anyof the compounds described above and a pharmaceutically acceptablecarrier. Another illustration of the invention is a process for making apharmaceutical composition comprising combining any of the compoundsdescribed above and a pharmaceutically acceptable carrier.

Another aspect of the present invention provides for the use of theshort nucleotide compounds and derivatives thereof and theirpharmaceutical compositions for the manufacture of a medicament for theinhibition of a susceptible viral infection in particular HCVreplication, and/or the treatment of one or more susceptible viralinfections, in particular HCV infection and/or HCV infection incombination with another viral infection such as HBV or HIV. Yet afurther aspect of the present invention provides for the shortoligonucleotide compounds and derivatives thereof and theirpharmaceutical compositions for use as a medicament for the inhibitionof RNA-dependent RNA viral replication, in particular HCV replication,and/or for the treatment of RNA-dependent RNA viral infection, inparticular HCV infection.

The pharmaceutical compositions of the present invention comprise atleast one compound of the invention as an active ingredient or apharmaceutically acceptable salt thereof, and may also contain apharmaceutically acceptable carrier and excipients and optionally othertherapeutic ingredients. By “pharmaceutically acceptable” is meant thatthe carrier, diluent, or excipient must be compatible with the otheringredients of the formulation and not deleterious to the recipientthereof. The compositions include compositions suitable for oral,rectal, topical, parenteral (including subcutaneous, intramuscular, andintravenous), ocular (ophthalmic), pulmonary (nasal or buccalinhalation), or nasal administration, although the most suitable routein any given case will depend on the nature and severity of theconditions being treated and on the nature of the active ingredient.They may be conveniently presented in unit dosage form and prepared byany of the methods well-known in the art of pharmacy.

In practical use, the compounds of the invention can be combined as theactive ingredient in intimate admixture with a pharmaceutical carrieraccording to conventional pharmaceutical compounding techniques. Thecarrier may take a wide variety of forms depending on the form ofpreparation desired for administration, e.g., oral or parenteral(including intravenous). In preparing the compositions for oral dosageform, any of the usual pharmaceutical media may be employed, such as,for example, water, glycols, oils, alcohols, flavoring agents,preservatives, coloring agents and the like in the case of oral liquidpreparations, such as, for example, suspensions, elixirs and solutions;or carriers such as starches, sugars, microcrystalline cellulose,diluents, granulating agents, lubricants, binders, disintegrating agentsand the like in the case of oral solid preparations such as, forexample, powders, hard and soft capsules and tablets, with the solidoral preparations being preferred over the liquid preparations.

Because of their ease of administration, tablets and capsules representthe most advantageous oral dosage unit form in which case solidpharmaceutical carriers are obviously employed. If desired, tablets maybe coated by standard aqueous or nonaqueous techniques. Suchcompositions and preparations should contain at least 0.1 percent ofactive compound. The percentage of active compound in these compositionsmay, of course, be varied and may conveniently be between about 2percent to about 60 percent of the weight of the unit. The amount ofactive compound in such therapeutically useful compositions is such thatan effective dosage will be obtained. The active compounds can also beadministered intranasally as, for example, liquid drops or spray.

The tablets, pills, capsules, and the like may also contain a bindersuch as gum tragacanth, acacia, corn starch or gelatin; excipients suchas dicalcium phosphate; a disintegrating agent such as corn starch,potato starch, alginic acid; a lubricant such as magnesium stearate; anda sweetening agent such as sucrose, lactose or saccharin. When a dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier such as a fatty oil. Various othermaterials may be present as coatings or to modify the physical form ofthe dosage unit. For instance, tablets may be coated with shellac, sugaror both. A syrup or elixir may contain, in addition to the activeingredient, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and a flavoring such as cherry or orange flavor.Various stabilizers may be added that would stabilize the activepharmaceutical ingredient against degradation, such as amino acids orpolyamines. Other excipients could include without limitation PEG 400,glycine, Vitamin E derivatives, Sorbitan mono-oleate, Chitosan, Cholinecitrate, Sorbitan monostearate, Tween 80, Igepal CA 630, Brij 35, NP-40and their analogous derivatives.

Compounds of the invention may also be administered parenterally.Solutions or suspensions of these active compounds can be prepared inwater suitably mixed with a surfactant such as hydroxy-propylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols and mixtures thereof in oils. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms. The pharmaceutical forms suitable forinjectable use include sterile aqueous solutions or dispersions andsterile powders for the extemporaneous reparation of sterile injectablesolutions or dispersions. In all cases, the form is preferably sterileand is preferably fluid to the extent that easy syringability exists. Itmust be stable under the conditions of manufacture and storage and mustbe preserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g. glycerol,propylene glycol and liquid polyethylene glycol), suitable mixturesthereof, and vegetable oils.

Any suitable route of administration may be employed for providing amammal, especially a human with a therapeutically effective dosage of acompound of the present invention. The terms “administration of” and“administering a” compound should be understood to mean providing acompound of the invention to the individual in need. According to themethods of treatment of the present invention, viral infections aretreated or prevented in a subject such as a human or lower mammal byadministering to the subject a therapeutically effective amount or aninhibitory amount of a compound of the present invention, in suchamounts and for such time as is necessary to achieve the desired result.An additional method of the present invention is the treatment ofbiological samples with an inhibitory amount of a compound ofcomposition of the present invention in such amounts and for such timeas is necessary to achieve the desired result.

The term “therapeutically effective amount” of a compound of theinvention, as used herein, means a sufficient amount of the compound soas to decrease the viral load in a biological sample or in a subject. Asis well understood in the medical arts, a therapeutically effectiveamount of a compound of this invention will be at a reasonablebenefit/risk ratio applicable to any medical treatment.

The term “inhibitory amount” of a compound of the present inventionmeans a sufficient amount to decrease the hepatitis C viral load in abiological sample or a subject. An inhibitory amount or dose of thecompounds of the present invention may range from about 0.1 mg/Kg toabout 500 mg/Kg, alternatively from about 1 to about 50 mg/Kg Inhibitoryamounts or doses will also vary depending on route of administration, aswell as the possibility of co-usage with other agents.

The term “biological sample(s),” as used herein, means a substance ofbiological origin intended for administration to a subject. Examples ofbiological samples include, but are not limited to, blood and componentsthereof such as plasma, platelets, subpopulations of blood cells and thelike; organs such as kidney, liver, heart, lung, and the like; sperm andova;

bone marrow and components thereof; or stem cells. Thus, anotherembodiment of the present invention is a method of treating a biologicalsample by contacting said biological sample with an inhibitory amount ofa compound or pharmaceutical composition of the present invention.

Upon improvement of a subject's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained when the symptomshave been alleviated to the desired level, treatment should cease. Thesubject may, however, require intermittent treatment on a long-termbasis upon any recurrence of disease symptoms.

It will be understood, however, that the total daily usage of thecompounds and compositions of the present invention will be decided bythe attending physician within the scope of sound medical judgment. Thespecific inhibitory dose for any particular patient will depend upon avariety of factors including the disorder being treated and the severityof the disorder; the activity of the specific compound employed; thespecific composition employed; the age, body weight, general health, sexand diet of the patient; the time of administration, route ofadministration, and rate of excretion of the specific compound employed;the duration of the treatment; drugs used in combination or coincidentalwith the specific compound employed; and like factors well known in themedical arts.

The total daily inhibitory dose of the compounds of this inventionadministered to a subject in single, multiple or in divided doses can bein amounts, for example, from 0.01 to 50 mg/kg body weight or moreusually from 0.1 to 25 mg/kg body weight. Single dose compositions maycontain such amounts or submultiples thereof to make up the daily dose.Multiple doses may be single doses taken at different time intervals. Ingeneral, treatment regimens according to the present invention compriseadministration to a patient in need of such treatment from about 10 mgto about 1000 mg of the compound(s) of this invention per day in singleor multiple doses.

Another aspect of the present invention comprises inhibiting or treatingHCV infection with a compound of the present invention in combinationwith one or more agents useful for treating HCV infection. Such agentsactive against HCV include, but are not limited to: ribavirin,levovirin, viramidine, thymosin alpha-1, interferon-β, interferon-α,pegylated interferon-α (peginterferon-α), a combination of interferon-αand ribavirin, a combination of peginterferon-α and ribavirin, acombination of interferon-α and levovirin, and a combination ofpeginterferon-α and levovirin. Interferon-α includes, but is not limitedto: recombinant interferon-α2a (such as Roferon interferon availablefrom Hoffmann-LaRoche, Nutley, N.J.), pegylated interferon-α2a(PEGASYS™), interferon-α2b (such as Intron-A interferon available fromSchering Corp., Kenilworth, N.J.), pegylated interferon-α2b(PEGINTRON™), a recombinant consensus interferon (such as interferonalphacon-1), and a purified interferon-α product. Amgen's recombinantconsensus interferon has the brand name INFERGEN®. Levovirin is theL-enantiomer of ribavirin which has shown immunomodulatory activitysimilar to ribavirin. Viramidine represents an analog of ribavirindisclosed in WO 01/60379 (assigned to ICN Pharmaceuticals). Inaccordance with this method of the present invention, the individualcomponents of the combination can be administered separately atdifferent times during the course of therapy or concurrently in dividedor single combination forms. The instant invention is therefore to beunderstood as embracing all such regimes of simultaneous or alternatingtreatment, and the term “administering” is to be interpretedaccordingly. It will be understood that the scope of combinations of thecompounds of this invention with other agents useful for treating HCVinfection includes in principle any combination with any pharmaceuticalcomposition for treating HCV infection or an HCV co infection withanother virus such as HBV or HIV. When a compound of the presentinvention or a pharmaceutically acceptable salt thereof is, used incombination with a second therapeutic agent active against HCV, the doseof each compound may be either the same as or different from the dosewhen the compound is used alone.

For the treatment of HCV infection, the compounds of the presentinvention may also be administered in combination with an agent that isan inhibitor of HCV NS3 serine protease. HCV NS3 serine protease is anessential viral enzyme and has been described to be an excellent targetfor inhibition of HCV replication. HCV NS3 protease as a target for thedevelopment of inhibitors of HCV replication and for the treatment ofHCV infection is discussed in B. W. Dymock, “Emerging therapies forhepatitis C virus infection,” Emerging Drugs, 6: 13-42 (2001).

Ribavirin, levovirin, and viramidine may exert their anti-HCV effects bymodulating intracellular pools of guanine nucleotides via inhibition ofthe intracellular enzyme inosine monophosphate dehydrogenase (IMPDH).IMPDH is the rate-limiting enzyme on the biosynthetic route in de novoguanine nucleotide biosynthesis. Ribavirin is readily phosphorylatedintracellularly and the monophosphate derivative is an inhibitor ofIMPDH. Thus, inhibition of IMPDH represents another useful target forthe discovery of inhibitors of HCV replication. Therefore, the compoundsof the present invention may also be administered in combination with aninhibitor of IMPDH, [see A. C. Allison and E. M. Eugui, Agents Action,44 (Suppl.): 165 (1993)].

For the treatment of HCV infection, the compounds of the presentinvention may also be administered in combination with the antiviralagent amantadine (1-aminoadamantane) [for a comprehensive description ofthis agent, see J. Kirschbaum, Anal. Profiles Drug Subs. 12: 1-36(1983)]. The compounds of the present invention may also be combined forthe treatment of HCV infection with antiviral 2′-C-branchedribonucleosides disclosed in R. E. Harry-O'kuru, et al., J. Org. Chem.,62: 1754-1759 (1997); M. S. Wolfe, et al., Tetrahedron Lett., 36:7611-7614 (1995). Such 2′-C-branched ribonucleosides include, but arenot limited to: 2′-C-methyl-cytidine, 2′-C-methyl-uridine,2′-C-methyl-adenosine, 2′-C-methyl-guanosine, and9-(2-C-methyl-β-D-ribofuranosyl)-2,6-diaminopurine. The compounds of thepresent invention may also be combined for the treatment of HCVinfection with nucleosides having anti-HCV properties. The compounds ofthe present invention may also be combined for the treatment of HCVinfection with non-nucleoside inhibitors of HCV polymerase.

Antiviral compounds which may be used in combination with the compoundsof the invention include but are not limited to lamivudine, adefovir,tenofovir, FTC, entecavir, acyclovir, pencyclovir, gancyclovir,interferons, pegylated interferons, ribavirin and other knowntherapeutic compositions for the treatment of viral infections and viralcoinfections such as HCV in combination with HIV and/or HBV.

The invention is further illustrated by the following non-limitingExamples.

Example 1 Synthesis of 3′-dApsU_(2′-OMe)

In the present studies, the R_(p),S_(p) mixture of the phosphorothioateanalog 3′-dApsU_(2′-OMe) as shown in Example 2, was synthesized in largescale (I millimol of nucleoside-loaded controlled-pore glass (CPG)support) using solid-phase phosphoramidite chemistry, (Beaucage, S. L.;Iyer, R. P. Tetrahedron 1993, 49, 1925) in conjunction with a speciallyfabricated LOTUS Reactor® (Padmanabhan, S.; Coughlin, J. E.; Iyer, R. P.Tetrahedron Lett. 2005, 46, 343; Iyer, R. P.; Coughlin, J. E.;Padmanabhan, S. Org. Prep. Proc. Intl. 2005, 37, 205). The dA-linked CPGsupport was prepared using an ultrafast functionalization and loadingprocess for solid supports. For the sulfurization of theinternucleotidic dinucleoside phosphite coupled product, a solution of3H-1,2-benzodithiole-3-one-1,1,-dioxide (0.4 M in dry CH₃CN) wasemployed (Iyer, R. P.; Regan, J. B.; Egan, W.; Beaucage, S. L. J. Am.Chem. Soc. 1990, 112, 1253). Removal of the 5′-trityl group wasaccomplished by a 2.5% solution of dichloroacetic acid indichloromethane. Removal of protecting groups at the base, and phosphateand cleavage of the nucleotide from the support was carried out bytreating the support-bound product with 28% ammonium hydroxide at roomtemperature for several hours. Following processing, chromatographicpurification, and lyophilization, the sodium salt of R_(p),S_(p)3′-dApsU_(2′-OMe) (˜60:40 mixture) was obtained >96% pure, which wascharacterized by ³¹P and ¹H NMR. ³¹P NMR, (D₂O), 57.0, and 57.7 ppm.

Alternatively, the title compound could also be prepared bysolution-phase method. Typically, 3′-protected 5′-hydroxy Nbz-dA wascontacted 5′-DMT-2′-OMe-3′-uridine phosphoramidite in the presence of anactivating agent such as tetrazole or thioethyl tetrazole in anhydrousdichloromethane or acetonitrile as solvent in an inert atmosphere.Examples of 3′-protected nucleoside include levulinyl,t-butyldimethylsilyl, benzoyl, 4-t-butylbenzoyl etc. The reactionmixture containing the dinucleoside monophosphite was quenched withwater and treated with a solution of a solution of3H-1,2-benzodithiole-3-one-1,1,-dioxide (0.4 M in dry CH₃CN) wasemployed (Iyer, R. P.; Regan, J. B.; Egan, W.; Beaucage, S. L. J. Am.Chem. Soc. 1990, 112, 1253). The reaction mixture was detritylated usingDCA/DCM and the solution diluted with water, and extracted withdichlormethan. The combined organic layers were washed with NaHCO3,water, and brine. The organic layer was concentrated to obtain the fullyprotected dinucleoside phosphotriester was processed. Detritylation andremoval of the protecting groups was accomplished as before. Followingprocessing, chromatographic purification, and lyophilization, the sodiumsalt of R_(p),S_(p) 5 (˜60:40 mixture) was obtained >96% pure asascertained by reversed-phase HPLC.

Other dinucleotides claimed in this invention can also be prepared usingsimilar procedures as above. It is pertinent to mention that thoseskilled in the art will be able to synthesize similar compounds usingthe classical phosphotriester approach or the H-phosphonate approach(Beaucage, S. L.; Iyer, R. P. Tetrahedron 1993, 49, 1925).

Example 2 S-isopropylcarbonyloxymethyl thiophosphate (6k) of3′dApsU_(2′OMe)

The target compound 6k (also designated Compound 2 in Table 1) isprepared in two steps.

Step 1. Preparation of Iodomethylisopropyl Carbonate.

To a solution of anhydrous sodium iodide (6 g, 40 mmol) in anhy.acetonitrile (20 mL) chloromethyl isopropyl carbonate (2.9 g, 19 mmol)in anhyd. acetonitrile (10 mL) was added dropwise over 20 min. Thereaction mixture, covered with aluminum foil (protected from light) wasstirred at room temperature overnight. The solid separated was filtered,washed with acetonitrile and the filtrate was concentrated under reducedpressure. Residue was dissolved in water (10 mL) and organics wereextracted in ether (25 mL). Ether extracts were washed with sodiumbisulfite (5%, 10 mL), later brine (10 mL). Organic layer was dried overanhd. sodium sulfate, filtered, concentrated and dried under high driedvacuum. Yield 2.72 g (58%); ¹H-NMR δ 1.3 (d, 6H), 4.95 (m, 1H), 5.95 (s,2H) ppm.

Step 2. Alkylation of Dinucleotide, 3′-ApsU2′OMe.

To a solution of dinucleotide of Example 1 (60 mg, 0.098 mmol) in water(HPLC, 400 mL) under stirring, a solution of iodomethyl isopropylcarbonate (80 mg, 0.0166 mmol, 3.33 eq) in acetone (1 mL) was added.

Additional acetone (1 mL) was added to get a clear solution to avoid anyseparation of oily globules of alkylating agent. The reaction mixture,covered in aluminum foil, was stirred for 3 h, concentrated underrotavap conditions and later in high vacuum to obtain the reactionmixture as a white solid. This was purified by silica columnchromatography using initially chloroform and slowly with chloroformcontaining 2% to finally 8% methanol. The fractions, containing majorcomponent, were combined, concentrated and dried under high vacuumovernight. The desired pure product 6k was isolated in almostquantitative yield (68 mg); ³¹P-NMR (MeOH-d₄) δ 27.7, 28.6.

Example 3 Preparation of S-Methyl Cholic Acid Ester 61 of3′dApsU_(2′OMe)

Step 1. Synthesis of Chloromethyl Deoxycholate.

To deoxycholic acid (120 mg, 0.306 mmol) in ethanol (4 mL) a solution ofcaesium carbonate (53 mg, 0.160 mmol) in water (3 mL) was added. Thereaction mixture was stirred for 30 min and ethanol was initiallyremoved under removed 61rotavap, and later under high vac. The residuewas lyophilized to give the cesium salt as white powder. To a solutionof cesium salt in N,N-dimethylformamide (DMF, 3 mL) at room temperaturebromochloromethane (10 mL) was added and the aluminum foil coveredreaction mixture was stirred at room temperature for 24 h. The solventswere removed and the reaction mixture was extracted in dichloromethane(20 mL), washed with water (5 mL), brine (5 mL) and solvent was removedafter drying over anhy. sodium sulfate to give the chloromethyl compound(100 mg, 74%). This was used without any further purification for theconversion to the corresponding iodomethyl derivative.

Step 2. Preparation of Iodomethyl Deoxycholate.

To a solution of sodium iodide (304 mg, 2.03 mmol) in anhyd.acetonitrile (3 mL) chloromethyl ester (438 mg, 0.99 mmol) in a mixtureof acetonitrile (6 mL) and dichloromethane (2 mL) was added slowly. Thereaction mixture, protection from light, was stirred at room temperatureover 48 hours. After concentration, the reaction mixture was extractedin dichloromethane (15 mL), organic layer was washed with water (5 mL),sodium bisulfite (5%, 5 mL) and finally brined (5 mL). Dried over anhyd.sodium sulfate and the crude product, obtained after removal of solvent,was purified by silica column chromatography to obtain the iodo compound(110 mg, 21%).

Step 3. Coupling of Iodomethyl Deoxycholate.

To a solution of 3′dApsU2′OMe (50 mg, 0.082 mmol) in water (400 mL) ofExample 1, a solution of iodomethyl deoxycholate (110 mg, 2.066 mmol) inacetone (3 mL) was added. The solid separated was dissolved by addingmore acetone (˜6 mL) and the reaction mixture was stirred overnight.Concentrated under vacuum and purified by silica column chromatographyusing chloroform to chloroform containing methanol (2 to 10%). Fractionswere combined, concentrated and dried under high vacuum to give thedesired product 6l (40 mg, 49%); ³¹P-NMR (MeOH) δ 28.2, 29.1 ppm.

Example 4 Preparation of N-(t-butoxycarbonyl)-L-phenylalaninate (6m)

Iodomethyl N-(t-butoxycarbonyl)-L-phenylalaninate. ToN-(t-butoxycarbonyl)-L-phenylglycine (663 mg, 2.49 mmol) in ethanol (3mL) a solution of cesium carbonate (427 mg, 1.31 mmol) in water (2 mL)was added. After the evolution of gas ceased, the reaction mixture wasstirred for 1 h. The solvents were removed and lyophilized to obtain thecesium salt. To a solution of cesium salt (270 mg, 0.82 mmol) inN,N-dimethylformamide (DMF, 2 mL) bromochloromethane (5 mL) was addedand stirred overnight with the reaction mixture covered with aluminumfoil. The solid separated was filtered, washed the solids with DMF (2mL), and the filtrate concentrated under high vacuum. The product (206mg, 80%) was found to be pure by TLC (Hex:EtOAc 4:1). This intermediatewas used for the conversion to iodo compound without furtherpurification. To a solution of sodium iodide (196 mg, 1.31 mmol) inanhyd. acetonitrile (3 mL), chloromethyl phenylalaniate derivative (206mg, 0.656 mmol) in anhyd. acetonitrile (1 mL) was added. The reactionmixture was stirred at room temperature, with protection from light,overnight. Filtered, washed the solid with DMF (3 mL), and concentratedthe filtrate under vacuum. The residue was extracted in dichloromethane(10 mL) and water (5 mL), washed the organic layer with NaHSO3 (5%, 5mL) and brine (satd., 5 mL). The organic layer was dried over anhyd.Na₂SO₄, and concentrated, to yield the desired iodo compound (199 mg,75%).

Alkylation of 3′dApsU_(2′OMe). To a solution of 3′dApsU2′OMe (44 mg,0.072 mmol) in water (400 ul) of Example 1, the iodide (100 mg, 0.25mmol) in acetone (800 ul) was added and the reaction mixture was stirredover night. The reaction mixture was concentrated under vacuum,lyophilized, and purified by silica column chromatography usingchloroform and mixture containing chloroform and methanol (2% to 10%).Fractions were collected, combined, concentrated and dried under highvacuum to give the t-Boc protected phenylalanine coupled product 6m (40mg, 65%); ³¹P-NMR (MeOH-d₄) δ 28.7, 27.9 ppm.

Example 5 Preparation of 4-acetamidobenzyl Derivative 6n of3′dApsU_(2′OMe)

Preparation of 4-acetamidobenzyl alcohol. To a solution of4-acetamidobenzaldehyde (10 g, 61.3 mmol) in methanol (100 mL) was addedsodium borohydride (800 mg) at room temperature in portions. Thereaction mixture was stirred over night, and the progress of reactionchecked by TLC using 4:1 hexanes:EtOAc as eluent. Absence of startingmaterial indicated the completion of reduction and the reaction mixturewas concentrated in a rotavap. The residue was partitioned between water(25 mL) and ethyl acetate (4×50 mL) and the organic layer was washedwith brine (25 mL). The ethyl acetate layer was dried over anhydroussodium sulfate and the removal of the solvent gave the alcohol as a paleyellow solid, which was dried under high vacuum. 8.6 g (85%); ¹H NMR(DMSO-d₆): δ 2.0 (s, 3H), 4.5 (d, 2H), 5.2 (t, 1H), 7.25 (d, 2H), 7.55(d, 2H), 9.95 (s, 1H) ppm.

Preparation of 4-acetamidobenzyl iodide. To a cooled solution of anhyd.DMF (5 mL) was added thionyl chloride (0.2 mL, 2.8 mmol). The mixturewas stirred for 10 min and a solution of KI (2.49 g, 15 mmol) in anhyd.DMF (12 mL) was added followed by the addition of the alcohol preparedabove (0.165 g, 1 mmol). The reaction mixture was stirred in theice-bath for 3 h and allowed to stir at r.t. overnight. The reactionmixture was poured into ice-water (25 mL) and extracted with ether (3×25mL). The ether layer was washed with brine, dried over anhyd. sodiumsulfate and concentrated to remove the solvent. The product was obtained(138 mg, 50%) as a clean yellow solid. (TLC Hex: EtOAc (1:1). ¹H NMR(CDCl₃): δ 2.17 (s, 3H), 4.45 (s, 2H), 7.17 (br.s, 1H), 7.33 (d, 2H),7.43 (d, 2H) ppm. This compound was also prepared with improved yields(˜75%) using cesium iodide and boron trifluoride etherate inacetonitrile. The coupling of 4-acetamidobenzyl iodide with 3′dApsU2′OMewas done as described for the cholic acid analog before.

Example 6 Synthesis of 4-benzamidobutyl Analog 6o of 3′dApsU_(2′OMe)

Preparation of 4-benzamidobutyl iodide. To cold anhydrous DMF (5 mL) at0-5° C. was added thionyl chloride (0.2 mL) and the mixture was stirredfor 15 min. A solution of potassium iodide (2.4 g, 5 mmol) in anhy. DMF(8 mL) followed by a solution of 4-benzamidobutanol (193 mg, 1 mmol) inanhy. DMF (2 mL) was added. The colored reaction mixture was stirredovernight. The reaction mixture was worked up by pouring into ice-coldwater (˜10 mL) and extracted with ether (3×15 mL). Finally, the etherlayer was washed with water, brine and dried over anhydrous sodiumsulfate. The crude product, obtained after filtration and removal of thesolvent, was purified by column chromatography using a mixture of hexaneand ethyl acetate (4:1) to give the iodo compound as an oil. 45%; ¹H NMR(CDCl₃): δ 1.77 (m, 2H), 1.93 (m, 2H), 3.23 (t, 2H), 3.55 (q, 2H), 6.26(br.s, 1H), 7.48 (m, 3H), 7.75 (m, 2H) ppm.

Coupling of the 4-benzamidobutyl iodide with 3′dApsU2′OMe was carriedout as before to obtain the title compound 6o.

Example 7 Synthesis of 5-benzoyloxypentyl Analog of 3′dApsU_(2′OMe)

Preparation of 5-benzoyloxypentan-1-ol. A mixture of benzoic acid (1 g),1,5-pentanediol (5 mL) and p-toluenesulfonic acid (110 mg) was heated inan oil-bath at 100° C. overnight. The reaction mixture was cooled toroom temperature, poured into water (50 mL) and extracted with EtOAc(2×25 mL), washed with sodium carbonated (5%, 20 mL) followed by brine(15 mL). The organic layer was dried over anhyd. sodium sulfate,filtered and concentrated to give almost pure product (1.15 g, 67%).

Preparation of 5-benzoyloxy-1-iodopentane. 36% yield. ¹H NMR (CDCl₃):

δ 1.57 (m, 2H), 1.85 (m, 4H), 3.22 (t, 2H), 4.33 (t, 2H), 7.44 (m, 2H),7.57 (m, 1H), 8.04 (m, 2H) ppm.

The coupling of 5-benzoyloxy-1-iodopentane with 3′dApsU2′OMe was carriedout as before.

Preparation of 5-benzoyloxybutan-1-ol. This was prepared in 73% yieldusing 1,4-butanediol in the procedure for 5-benzoylpentan-1-ol.

Example 8 Synthesis of 4-acetoxybenzyl Analog 6q of 3′dApsU_(2′OMe)

Step 1. Preparation of 4-acetoxybenzyl alcohol. To a cooled suspensionof 4-hydroxybenzyl alcohol (1.95 g, 14 mmol) in ethyl acetate (25 mL) inan ice-bath, triethylamine (2.1 mL, 14.9 mmol) was added in one lotunder stirring. A solution of acetyl chloride (1.1 mL, 15.5 mmol) inethyl acetate (12 mL) was added dropwise from an addition funnel. Thereaction mixture was stirred overnight. The solid was filtered, washedwith ethyl acetate and the residue, after concentration, was purified bycolumn chromatography using hexanes initially and later gradually to 40%ethyl acetate. Yield 40%. ¹H-NMR (CDCl₃), δ 2.02 (br. s, 1H), 2.29 (s,3H), 4.65 (s, 2H), 7.07 (d, 2H), 7.36 (d, 2H) ppm.

Step 2. Preparation of 4-acetoxybenzyl iodide. To a solution of4-acetoxybenzyl alcohol (0.332 g, 2 mmol), and cesium iodide (0.571 g,2.2 mmol) in anhyd. acetonitrile (10 mL) under nitrogen, borontrifluoride etherate (0.28 mL, 2.2 mmol) in acetonitrile (5 mL) wasintroduced. After stirring overnight, the reaction mixture was pouredinto ice-cold water (20 mL) and the solid separated was filtered, washedwith water and later with hexanes. The product was dried under highvacuum. Yield, 0.39 g, 71%; TLC, hexanes:EtOAC (4:1). ¹H NMR (CDCl₃): δ2.3 (s, 3H), 4.35 (s, 2H), 7.05 (d, 2H), 7.5 (d, 2H) ppm.

Step 3. Synthesis of 4-acetoxybenzyl analog of 3′dApsU_(2′OMe).Alkylation of 3′dApsU_(2′OMe) with 4-acetoxybenzyl iodide was carriedout as before.

Example 9 Primary and Secondary Anti-HCV Assays

Primary Anti-HCV Assay—

Antiviral activity against HCV was assessed in a 3-day assay (Okuse, etal., 2005; Antiviral. Res. 65:23; Korba, et al., 2008, Antiviral Res.77:56) using the stably-expressing HCV replicon cell line, AVA5[sub-genomic (CON1), genotype 1b] (Blight, et al., 2000, Science290:1972) maintained as sub-confluent cultures on 96-well plates.Antiviral activity was determined by blot hybridization analysis ofintracellular HCV RNA (normalized to the level of cellular B-actin RNAin each culture sample). Cytotoxicity was assessed by neutral red dyeuptake in cultures maintained in parallel plates.

EC₅₀, EC₉₀, and CC₅₀ values were calculated by linear regressionanalysis (MS EXCEL®, QUATTROPRO®) using data combined from all treatedcultures Korba & Gerin, 1992, Antivir. Res. 19:55; Okuse, et al., 2005,Antivir. Res. 65:23). Standard deviations for EC₅₀ and EC₉₀ values werecalculated from the standard errors generated by the regressionanalyses. EC₅₀ and EC₉₀ were drug concentrations at which a 2-fold, or a10-fold depression of intracellular HCV RNA (relative to the averagelevels in untreated cultures), respectively was observed. CC₅₀ was thedrug concentration at which a 2-fold lower level of neutral red dyeuptake (relative to the average levels in untreated cultures) wasobserved. The Selectivity index (S.I.) was calculated as CC₅₀/EC₅₀.Recombinant human interferon 2b (PBL laboratories, Inc.) was used as anassay control.

Secondary Anti-HCV Assay—

This assay assesses activity against different HCV genotypes using theformat described for the primary assay. Activity against the genotype 1bHCV is included for comparison. Currently available is a replicon cellline containing H/FL-Neo (genotype 1a (H77), full length construct)(Blight, et al., 2003, J. Virol. 77:3181). Replicon cell line AVA5(sub-genomic (CON1), genotype 1b; (Blight, et al., 2000, Science290:1972).

The results of the primary and secondary assays are shown in Table 3.

TABLE 3 EC₅₀ EC₅₀ EC₉₀ EC₉₀ HCV Type HCV type HCV type HCV type Compd 1A1B 1A 1B CC₅₀ # Structure micromolar micromolar micromolar micrmolarmicromolar 2

2.2 1 8   6   >100 1

2.9 NA 8.5 NA >100 Interferon (alfNB2)/mL (positive control 1.8 2 8  8.5 >100

Example 10 Cytotoxicity Assays

Cellular cytotoxicity profile of the compounds was carried out against apanel of cell lines. Standard MTT assays were performed in 96-wellplates using the Promega CellTiter96 Non-radioactive Cell ProliferationAssay Kit in conjunction with a 96-well Plate Reader (ThermoMax,Molecular devices), and MDBK, Vero, and HFF cell lines (obtained fromATCC). Several controls were employed including the nucleoside analogs3TC, AZT, and ddC, as well as, media without drugs. SDS was used as apositive cytotoxic control. The compounds were tested in triplicate atconcentrations of 100, 300, and 1000 μM. Following a 24-hour incubationof cells with the test substance, the MTT assay was carried out. Thedata is shown in Table 4.

TABLE 4 In vitro cytotoxicity studies of compounds in various cell linesMDBK HFF HepG2 Compound Vero (CC₅₀, (CC₅₀, (CC₅₀, # Compound (CC₅₀, uM)uM) uM) uM) 2

>1000 >1000 >1000 >100 1

>1000 >1000 >1000 >100

Example 11 Inhibition Assays

The effectiveness of the compounds of the present invention asinhibitors of HCV NS5B RNA-dependent RNA polymerase (RdRp) can bemeasured in the following assay.

Assay for Inhibition of HCV NS5B Polymerase

This assay is used to measure the ability of the compounds of thepresent invention to inhibit the enzymatic activity of the RNA-dependentRNA polymerase (NS5B) of the hepatitis C virus (HCV) on a heteromericRNA template.

Procedure: Assay Buffer Conditions:

(50 μL-total/reaction) 20 mM Tris, pH 7.5 50 μM EDTA 5 mM DTT 2 mM MgCl₂80 mM KCl 0.4 U/μL RNAsin (Promega, stock is 40 units/μL) 0.75 μg t500(a 500-nt RNA made using T7 runoff transcription with a sequence fromthe NS2/3 region of the hepatitis C genome) 1.6 μg purified hepatitis CNS5B (form with 21 amino acids C-terminally truncated) 1 μM A,C,U,GTP(Nucleoside triphosphate mix) [alpha-³³P]-GTP or [alpha³²P]-GTP.

The compounds are tested at various concentrations up to 100 μM finalconcentration.

An appropriate volume of reaction buffer is made including enzyme andtemplate t500. Thionucleoside derivatives of the present invention arepipetted into the wells of a 96-well plate. A mixture of nucleosidetriphosphates (NTP's), including the radiolabeled GTP, is made andpipetted into the wells of a 96-well plate. The reaction is initiated byaddition of the enzyme-template reaction solution and allowed to proceedat room temperature for 1-2 h.

The reaction is quenched by addition of 20 μL 0.5M EDTA, pH 8.0. Blankreactions in which the quench solution is added to the NTPs prior to theaddition of the reaction buffer were included.

50 μL of the quenched reaction are spotted onto DE81 filter disks(Whatman) and allowed to dry for 30 min. The filters are washed with 0.3M ammonium formate, pH 8 (150 mL/wash until the cpm in 1 mL wash is lessthan 100, usually 6 washes). The filters are counted in 5-mLscintillation fluid in a scintillation counter.

The percentage of inhibition is calculated according to the followingequation: % Inhibition=[1−(cpm in test reaction−cpm in blank)/(cpm incontrol reaction−cpm in blank)]×100.

Counterscreens

The ability of the compounds of the present invention to inhibit humanDNA polymerases are measured in the following assays.

a. Inhibition of Human DNA Polymerases Alpha and Beta:

Reaction Conditions:

50 μL reaction volume Reaction Buffer Components: 20 mM Tris-HCl, pH 7.5200 μg/mL bovine serum albumin 100 mM KCl 2 mM β-mercaptoethanol 10 mMMgCl₂ 1.6 μM dA, dG, dC, dTTP α³³P-dATp Enzyme and Template: 0.05 mg/mLgapped fish sperm DNA template 0.01 U/μL DNA polymerase α or βPreparation of Gapped Fish Sperm DNA Template: Add 5 μL 1M MgCl₂ to 500μL activated fish sperm DNA (USB 70076); Warm to 37° C. and add 30 μL of65 U/μL of exonuclease III (GibcoBRL 18013-011); Incubate 5 min at 37°C.; Terminate reaction by heating to 65° C. for 10 min; Load 50-100 μLaliquots onto Bio-spin 6 chromatography columns (Bio-Rad 732-6002)equilibrated with 20 mM Tris-HCl, pH 7.5; Elute by centrifugation at1,000.times.g for 4 min; Pool eluate and measure absorbance at 260 nm todetermine concentration.

The DNA template is diluted into an appropriate volume of 20 mMTris-HCl, pH 7.5 and the enzyme is diluted into an appropriate volume of20 mM Tris-HCl, containing 2 mM β-mercaptoethanol, and 100 mM KCl.Template and enzyme are pipetted into microcentrifuge tubes or a 96 wellplate. Blank reactions excluding enzyme and control reactions excludingtest compound are also prepared using enzyme dilution buffer and testcompound solvent, respectively. The reaction is initiated with reactionbuffer with components as listed above. The reaction wars incubated for1 hour at 37° C. The reaction was quenched by the addition of 20 μL 0.5MEDTA. 50 μL of the quenched reaction is spotted onto Whatman DE81 filterdisks and air dried. The filter disks are repeatedly washed with 150 mL0.3M ammonium formate, pH 8 until 1 mL of wash is <100 cpm. The disksare washed twice with 150 mL absolute ethanol and once with 150 mLanhydrous ether, dried and counted in 5 mL scintillation fluid.

The percentage of inhibition is calculated according to the followingequation: % inhibition=[1−(cpm in test reaction−cpm in blank)/(cpm incontrol reaction−cpm in blank)]×100.

Inhibition of Human DNA Polymerase Gamma:

The potential for inhibition of human DNA polymerase gamma is measuredin reactions that included 0.5 ng/μL enzyme; 10 μM DATP, dGTP, dCTP, andTTP; 2 μCi/reaction [α³³P]-dATP, and 0.4 μg/μL activated fish sperm DNApurchased from US Biochemical) in a buffer containing 20 mM Tris pH8, 2mM β-mercaptoethanol, 50 mM KCl, 10 mM MgCl₂, and 0.1 μg/μL BSA.Reactions are allowed to proceed for 1 h at 37° C. and were quenched byaddition of 0.5 M EDTA to a final concentration of 142 mM. Productformation is quantified by anion exchange filter binding andscintillation counting. Compounds were tested at up to 50 μM.

The percentage of inhibition was calculated according to the followingequation: % inhibition=[1−(cpm in test reaction−cpm in blank)/(cpm incontrol reaction−cpm in blank)]×100.

The ability of the compounds of the present invention to inhibit HIVinfectivity and HIV spread is measured in the following assays.

HIV Infectivity Assay:

Assays are performed with a variant of HeLa Magi cells expressing bothCXCR4 and CCR5 selected for low background β-galactosidase (β-gal)expression. Cells are infected for 48 h, and β-gal production from theintegrated HIV-1 LTR promoter was quantified with a chemiluminescentsubstrate (Galactolight Plus, Tropix, Bedford, Mass.). Inhibitors weretitrated (in duplicate) in twofold serial dilutions starting at 100 μM;percent inhibition at each concentration is calculated in relation tothe control infection.

Inhibition of HIV Spread:

The ability of the compounds of the present invention to inhibit thespread of the human immunedeficiency virus (HIV) is measured by themethod described in U.S. Pat. No. 5,413,999 (May 9, 1995), and J. P.Vacca, et al., Proc. Natl. Acad. Sci., 91: 4096-4100 (1994), which areincorporated by reference herein in their entirety.

The following assays were employed to measure the activity of thecompounds of the present invention against other RNA-dependent RNAviruses: Determination of In Vitro Antiviral Activity of CompoundsAgainst Rhinovirus (Cytopathic Effect Inhibition Assay); assayconditions are described in the article by Sidewall and Huffman, “Use ofdisposable microtissue culture plates for antiviral and interferoninduction studies,” Appl. Microbiol. 22: 797-801 (1971).

Viruses:

Rhinovirus type 2 (RV-2), strain HGP, is used with KB cells and media(0.1% NaHCO₃, no antibiotics) as stated in the Sidwell and Huffmanreference. The virus, obtained from the ATCC, is from a throat swab ofan adult male with a mild acute febrile upper respiratory illness.

Rhinovirus type 9 (RV-9), strain 211, and rhinovirus type 14 (RV-14),strain Tow, are also obtained from the American Type Culture Collection(ATCC) in Rockville, Md. RV-9 is from human throat washings and RV-14 isfrom a throat swab of a young adult with upper respiratory illness. Bothof these viruses are used with HeLa Ohio-1 cells (Dr. Fred Hayden, Univ.of VA) which are human cervical epitheloid carcinoma cells. MEM (Eagle'sminimum essential medium) with 5% Fetal Bovine serum (FBS) and 0.1%NaHCO₃ is used as the growth medium.

Antiviral test medium for all three virus types was MEM with 5% FBS,0.1% NaHCO₃, 50 μg gentamicin/mL, and 10 mM MgCl₂.

2000 μg/mL is the highest concentration used to assay the compounds ofthe present invention. Virus is added to the assay plate approximately 5min after the test compound. Proper controls are also run. Assay platesare incubated with humidified air and 5% CO₂ at 37° C. Cytotoxicity ismonitored in the control cells microscopically for morphologic changes.Regression analysis of the virus CPE data and the toxicity control datagave the ED50 (50% effective dose) and CC50 (50% cytotoxicconcentration). The selectivity index (SI) is calculated by the formula:SI=CC50/ED50.

Determination of In Vitro Antiviral Activity of Compounds AgainstDengue, Banzi, and Yellow Fever (CPE Inhibition Assay.

Assay details are provided in the Sidewall and Huffman reference above.Viruses:

Dengue virus type 2, New Guinea strain, is obtained from the Center forDisease Control. Two lines of African green monkey kidney cells are usedto culture the virus (Vero) and to perform antiviral testing (MA-104).Both Yellow fever virus, 17D strain, prepared from infected mouse brain,and Banzi virus, H 336 strain, isolated from the serum of a febrile boyin South Africa, are obtained from ATCC. Vero cells are used with bothof these viruses and for assay.

Cells and Media:

MA-104 cells (BioWhittaker, Inc., Walkersville, Md.) and Vero cells(ATCC) are used in Medium 199 with 5% FBS and 0.1% NaHCO₃ and withoutantibiotics. Assay medium for dengue, yellow fever, and Banzi viruses isMEM, 2% FBS, 0.18% NaHCO₃ and 50 μg gentamicin/mL.

Antiviral testing of the compounds of the present invention is performedaccording to the Sidewall and Huffman reference and similar to the aboverhinovirus antiviral testing. Adequate cytopathic effect (CPE) readingsare achieved after 5-6 days for each of these viruses.

Determination of In Vitro Antiviral Activity of Compounds Against WestNile Virus (CPE Inhibition Assay).

Assay details are provided in the Sidewall and Huffman reference citedabove. West Nile virus, New York isolate derived from crow brain, isobtained from the Center for Disease Control. Vero cells are grown andused as described above. Test medium is MEM, 1% FBS, 0.1% NaHCO₃ and 50μg gentamnicin/mL.

Antiviral testing of the compounds of the present invention is performedfollowing the methods of Sidewall and Huffman, which are similar tothose used to assay for rhinovirus activity. Adequate cytopathic effect(CPE) readings are achieved after 5-6 days. Determination of In VitroAntiviral Activity of Compounds Against Rhino, Yellow Fever, Dengue,Banzi, and West Nile Viruses (Neutral Red Uptake Assay)

After performing the CPE inhibition assays above, an additionalcytopathic detection method is used which is described in “MicrotiterAssay for Interferon: Microspectrophotometric Quantitation of CytopathicEffect,” Appl. Environ. Microbiol. 31: 35-38 (1976). A Model EL309microplate reader (Bio-Tek Instruments Inc.) is used to read the assayplate. ED50's and CD50's were calculated as above.

Example 12 Synergistic Antiviral Activity of Compound 2 in Combinationwith the Anti-HCV Compounds, Ribavirin, Interferon, Nucleoside Analog2′CmeC and Protease Inhibitor VX-950

Combination treatments of Compound 2 of Table 1 (also referred to in theschemes as 6k) with interferon, ribavirin, VX-950 (protease inhibitor),and 2′CmeCyt (polymerase inhibitor) were carried out using the primaryreplicon assay as described in Example 9. Briefly, two agents were mixedtogether at a predetermined relative ratios of the individual agentsbased on the EC₉₀ values of each compound (drug concentrations at whicha 10-fold reduction of HCV RNA is observed). For each combination ofagents, three concentration ratios, centered on the use of the compoundsat equipotent antiviral concentrations, were used. A dilution series(six three-fold-concentration steps, beginning at the approximate EC₉₀)was then generated with the concentration ratio of the two agentsremaining the same in each dilution step. Toxicity analysis wasperformed as described above for the monotherapies. Analysis of druginteractions in the combination studies was determined by the use of theCALCUSYN program (Biosoft, Inc., Cambridge, United Kingdom). Thisprogram evaluates synergy, additivity, or antagonism by use of severalmethodologies, including that of Chou and Talalay with a statisticalanalysis employing the Monte Carlo technique to provide confidencelimits, fraction-affected-confidence interval (FA-CI) plots,isobolograms, and median-effect plots. (Belenkii, M. S. Schinazi, R. Amethod for the analysis of combination therapies with statisticalanalysis. Antiviral Res. 25, 11, 2005). The data is shown in Table 5.

TABLE 5 Control 2 (μM) Geno- Control 1: alFNB2(IU/mL) Control 2 Cmpd #type CC50 EC50 EC90 SI CC50 EC50 EC90 SI Drug CC50 EC50 EC90 SI Comment2 (6k) 1B >100 >2.3 8.9 >43 >10000 2.1 6.7 >4762 2′CmeCyt >300 1.45.3 >214 2 + ribavirin 1B >100 >2.3 8.8 >43 >10000 2.1 6.7 >47622′CmeCyt >300 1.4 5.3 >214 synergistic interaction 2 + interferon @1B >100 >0.643 2.9 >156 >10000 2.1 6.7 >4762 2′CmeCyt >300 1.4 5.3 >214synergistic 1:1 interaction 2 + interferon @ 1B >100 >0.6292.6 >159 >10000 2.1 6.7 >4762 2′CmeCyt >300 1.4 5.3 >214 synergistic1:1 + 30 uM interaction ribavirin 2 + interferon @ 1B >100 >0.6582.8 >152 >10000 2.1 6.7 >4762 2′CmeCyt >300 1.4 5.3 >214 synergistic 1:3interaction 2 + interferon @ 1B >100 >0.686 2.5 >146 >10000 2.16.7 >4762 2′CmeCyt >300 1.4 5.3 >214 synergistic 1:3 + 30 uM interactionribavirin 2 + interferon @ 1B >100 >0.662 2.8 >151 >10000 2.1 6.7 >47622′CmeCyt >300 1.4 5.3 >214 synergistic 3:1 interaction 2 + interferon @1B >100 >0.777 2.7 >129 >10000 2.1 6.7 >4762 2′CmeCyt >300 1.4 5.3 >214synergistic 3:1 + 30 uM interaction ribavirin 2 + 2′CmeC@1:11B >100 >0.494 1.8 >202 >10000 2.1 6.7 >4762 2′CmeCyt >300 1.4 5.3 >214synergistic interaction 2 + 2′CmeC@1:3 1B >100 >0.462 1.5 >216 >100002.1 6.7 >4762 2′CmeCyt >300 1.4 5.3 >214 synergistic interaction 2 +2′CmeC@3:1 1B >100 >0.552 1.3 >192 >10000 2.1 6.7 >4762 2′CmeCyt >3001.4 5.3 >214 synergistic interaction 2 + VX-950@10:1 1B >100 >0.1180.928 >847 >10000 2.1 6.7 >4762 VX-950 88 0.249 0.892 353 synergisticinteraction 2 + VX-950@100:1 1B >100 >0.108 0.952 >926 >10000 2.16.7 >4762 VX-950 88 0.249 0.892 353 synergistic interaction 2 +VX-950@30:1 1B >100 >0.126 0.996 >794 >10000 2.1 6.7 >4762 VX-950 880.249 0.892 353 synergistic interaction

Example 13 In Vitro Bacterial Mutagenicity Assay of the Compound 2

Procedures: Compound 2 was formulated in desiccated dimethylsulfoxide(DMSO) and tested at a maximum concentration of 5000 μg/plate (thestandard limit dose for this assay) together with an appropriate numberof half-log₁₀ dilutions using the pre-incubation version of thebacterial mutation test. The absence of colonies on sterility checkplates confirmed absence of microbial contamination. The mean revertantcolony counts for the vehicle controls were close to or within thelaboratory historical control range.

Dispense 100 microliter aliquots of the appropriate bacterial culturesinto sample tubes stored on ice. Dispense aliquots of the vehicle,positive controls or dose formulations into the appropriate sampletubes. 2 mL top Agar and HBT cap was added to the sample tube, invertedthree times and poured into MG plate. The lid was replaced on the plateand left on a level surface to let the agar harden. The plates wereincubated in an incubator for 48 to 72 h. The background lawn and countthe number of revertants was checked for each plate.

Controls:

Appropriate positive control compounds (with S9 mix where required)induced increases in revertant colony numbers to at least twice theconcurrent vehicle control levels with the appropriate bacterial strain(1.5×for strain TA100), confirming the sensitivity of the test systemand activity of the S9 mix.

Results:

No visible thinning of the background lawn of non-revertant bacteria wasobtained following exposure to Compound 2, indicating that the testarticle was non-toxic to the bacteria at the levels tested. Noprecipitation was observed.

No substantial increases in the revertant colony counts were obtainedwith any strain following exposure to the test article Compound 2 ineither the absence or presence of S9 mix. It is therefore concluded thatCompound 2 did not show any evidence of genotoxic activity in this invitro mutagenicity assay. The data is shown in Tables 6, 7, 8, 9, and10, below.

TABLE 6 Compound 2 Tested in the Absence of S9 Conc. Plate ob- Fold (μg/Number of revertants servations* re- Strain plate) S9 x₁ x₂ x₃ mean SDx₁ x₂ x₃ sponse† TA1535 DMSO 0 26 23 29 26 3 1.0 50 0 10 15 16 14 3  0.5A 158 0 21 26 21 23 3 0.9 500 0 15 20 17 17 3 0.7 1581 0 13 18 17 16 30.6 5000 0 18 18 17 18 1 0.7 TA1537 DMSO 0 16 15 8 13 4 1.0 50 0 8 17 911 5 0.9 158 0 9 8 19 12 6 0.9 500 0 15 6 10 10 5 0.8 1581 0 17 15 13 152 1.2 5000 0 15 9 8 11 4 0.8 TA98 DMSO 0 31 27 35 31 4 1.0 50 0 18 15 3322 10 0.7 158 0 28 28 25 27 2 0.9 500 0 21 20 31 24 6 0.8 1581 0 24 1833 25 8 0.8 5000 0 27 26 20 24 4 0.8 *Comments on the plate orbackground lawn if applicable: contamination (C), incomplete lawn (IL),no lawn (NL), not required (NR), poor lawn (PL), precipitate (ppt) †Foldresponse in mean revertants compared to concurrent vehicle control SDSample standard deviation (note that SDs based on two values may beunreliable) A Apparent decreases in colony counts considered to be dueto normal variation rather than toxicity since not dose-related and notoutside historical control range

TABLE 7 Compound 2 Tested in the Absence of S9 Plate ob- Conc. Number ofrevertants servations* Fold Strain (μg/plate) S9 x₁ x₂ x₃ mean SD x₁ x₂x₃ response† TA100 DMSO 0 101 126 106 111 13 1.0 50 0 98 112 127 112 151.0 158 0 110 133 119 121 12 1.1 500 0 134 127 138 133 6 1.2 1581 0 122107 109 113 8 1.0 5000 0 98 126 122 115 15 1.0 WP2 uvrA DMSO 0 31 43 4038 6 1.0 50 0 34 43 45 41 6 1.1 158 0 49 44 33 42 8 1.1 500 0 39 26 3834 7 0.9 1581 0 44 56 51 50 6 1.3 5000 0 62 39 54 52 12 1.4 *Comments onthe plate or background lawn if applicable: contamination (C),incomplete lawn (IL), no lawn (NL), not required (NR), poor lawn (PL),precipitate (ppt). †Fold response in mean revertants compared toconcurrent vehicle control

TABLE 8 Compound 2 - Tested in the Presence of S9 Conc. Plate ob- Fold(μg/ Number of revertents servations* re- Strain plate) S9 x₁ x₂ x₃ meanSD x₁ x₂ x₃ sponse† TA1535 DMSO + 22 18 29 23 6 1.0 50 + 21 15 20 19 30.8 158 + 11 20 16 16 5 0.7 500 + 19 13 20 17 4 0.8 1581 + 27 21 17 22 50.9 5000 + 18 16 16 17 1 0.7 TA1537 DMSO + 16 23 18 19 4 1.0 50 + 20 1715 17 3 0.9 158 + 16 18 21 18 3 1.0 500 + 25 12 15 17 7 0.9 1581 + 18 1513 15 3 0.8 5000 + 11 15 10 12 3 0.6 TA98 DMSO + 41 52 45 46 6 1.0 50 +43 39 53 45 7 1.0 158 + 37 48 34 40 7 0.9 500 + 31 40 46 39 8 0.8 1581 +44 44 46 45 1 1.0 5000 + 53 36 43 44 9 1.0 *Comments on the plate orbackground lawn if applicable: contamination (C), incomplete lawn (IL),no lawn (NL), not required (NR), poor lawn (PL), precipitate (ppt) †Foldresponse in mean revertants compared to concurrent vehicle control SDSample standard deviation (note that SDs based on two values may beunreliable)

TABLE 9 Compound 2- Tested in the Absence of S9 Plate ob- Conc. Numberof revertants servations* Fold Strain (μg/plate) S9 x₁ x₂ x₃ mean SD x₁x₂ x₃ response† TA1535 DMSO + 163 153 113 143 26 1.0 50 + 111 109 100107 6 0.7 158 + 115 133 111 120 12 0.8 500 + 134 137 108 126 16 0.91581 + 136 122 121 126 8 0.9 5000 + 122 130 126 126 4 0.9 WP2 uvrADMSO + 37 35 44 39 5 1.0 50 + 48 43 42 44 3 1.1 158 + 46 43 40 43 3 1.1500 + 36 46 40 41 5 1.1 1581 + 52 43 45 47 5 1.2 5000 + 61 60 65 62 31.6 *Comments on the plate or background lawn if applicable:contamination (C), incomplete lawn (IL), no lawn (NL), not required(NR), poor lawn (PL), precipitate (ppt) †Fold response in meanrevertants compared to concurrent vehicle control SD Sample standarddeviation (note that SDs based on two values may be unreliable)

TABLE 10 Positive Controls for the Test Conc. Number of revertants FoldStrain Treatment (μg/plate) S9 x₁ x₂ x₃ mean SD response† TA1535 NaAz0.5 0 340 312 312 321 16 12 TA1537 9AC 10 0 728 910 585 741 163 57 TA982NF 1 0 151 114 153 139 22 4.5 TA100 NaAz 0.5 0 568 569 543 560 15 5.0WP2 uvrA NQO 0.5 0 1090 1277 1300 1222 115 32 TA1535 2AA 5 + 263 242 257254 11 11 TA1537 BaP 5 + 112 120 160 131 26 7 TA98 BaP 5 + 267 336 377327 56 7 TA100 BaP 5 + 724 910 882 839 100 6.0 WP2 uvrA 2AA 15 + 476 357285 373 96 9.6 †Fold response in mean revertants compared to concurrentvehicle control SD Sample standard deviation (note that SDs based on twovalues may be unreliable) All positive controls are known mutagens

Example 14 Tolerability Study of Compound 2 in uPA SCID (KMT) Mouse

The goals of the current study were to evaluate the tolerability ofCompound 2 (drug) in KMT mice over a 14 day dosing period and todetermine the pharmacokinetic properties of the drug in the studyanimals. Two separate groups of animals were treated with vehiclecontrol or with a daily drug dose of 300 mg/kg for 14 days. Bloodsamples were taken 15 minutes after the drug doses on Day 1 (firstdose), Day 7 and Day 14 (last dose) for measurement of the concentrationof the drug in the serum of study animals. Body weights and health indexscores were monitored daily in order to assess tolerance of the studyanimals to the dosing procedure and the drug. On each morning of thestudy one bottle of Compound 2 was dissolved in the 0.05 M citric acid,pH 3.0, vehicle to the required concentration of 40 mg/mL. The solutionwas used to dose animals in a volume of 7.5 ml/kg. The citric acidbuffer was also used for dosing of the vehicle control animals.

Ten KMT transgenic animals with low engraftment of human liver cells(hAAT values less than 20 at 6 weeks) were allocated to this study.Individual body weights of animals were recorded prior to treatment anddaily during the study. Animals were also evaluated once a day (atmorning dose administration) for clinical signs and monitored formorbidity and mortality. The study animals were allocated into 2 groupsof 5 animals. Compound 2 or citric acid vehicle control wereadministered once a day by oral gavage employing a sterile syringe andfeeding needle for each animal. Dosing volume was 7.5 mL/kg (150 μL/20grams body weight) for all groups. The volume was adjusted based on thedaily body weight of each individual animal. Group 1 animals receivedthe citric acid control and Group 2 animals received Compound 2 at 300mg/kg. The study started on Day 1 with the first drug dose administeredin the morning and continued up to Day 14, the last day of drugtreatment. Blood was collected via the central tail artery from allanimals of each group on Day 1, fifteen minutes after the first drugdose, on Day 7, fifteen minutes after the drug dose, and on Day 14,fifteen minutes after the last drug dose. The blood samples were allowedto clot and the serum removed from above the clot pellet. Serum sampleswere stored at −80° C. prior to shipment to the study client. The finalexperimental schedule is summarized below in Table 11.

TABLE 11 Experimental schedule for study NEA-1 Blood Animal VolumeID/Group Day(s) Date Manipulation Required 1 - 2 1 Dosing of animals n/a1 - 2 1 + 15 min. 15 minute PK blood draw 100 μl 1 - 2 2-7 Dosing ofanimals n/a 1 - 2 7 + 15 min. 15 minute PK blood draw 100 μl 1 - 2 8-14Dosing of animals n/a 1 - 2 14 + 15 min. 15 minute PK blood draw 100 μl

Individual body weights of the animals were recorded prior to treatmentand daily during the course of the study. The animals were alsoevaluated daily for clinical signs and monitored for morbidity andmortality. The standardized scale used to monitor morbidity (healthindex) of study animals is summarized below in Table 12.

TABLE 12 Mouse morbidity indexing scale Index Attributes 1 Normal shinycoat, bright eyes, very active, good body condition, grooming, normalbehaviour, normal eating/drinking pattern 1-2 Slightly scruffy coat,dull coat, greasy coat, less active 2 As above plus slight lethargy,slight dehydration, sunken eyes 2-3 As above plus increased scruffiness,dehydrated, emaciated, lethargic, slight hunched posture 3 As above plusextreme non-grooming (terrible coat appearance) bony along spine,hunched posture, emaciated, diarrhea 3-4 As above plus moribund,requiring euthanasia 4 Euthanized or found dead

Compound 2 was generally well tolerated by the study animals. One animalin the vehicle control group exhibited a moderately elevated healthindex from Day 11 thru Day 14 of the study. Two animals in the drugtreatment group also exhibited moderately elevated health index values.All other study animals completed the study with health values that wereequal to or half a score greater than their initial values.

Health index values are shown in Table 13.

TABLE 13 Health index values for individual animals in all treatmentgroups. Group ID # D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12 D13 D14 Group1 F139 1-2 1-2 1-2 1-2 1-2 1-2 2 2 1-2 1-2 2 2-3 2-3 2-3 Citrate ControlF146 1-2 1-2 1-2 1-2 1-2 1-2 1-2 1-2 1-2 1-2 1-2 1-2 1-2 1-2 F163 1-2 11-2 1-2 1-2 1-2 1-2 1-2 1-2 1-2 1-2 1-2 1-2 1-2 F170 1 1 1-2 1-2 1-2 1-21-2 1-2 1-2 1-2 1-2 1-2 1-2 1-2 F189 1 1-2 2 1-2 1-2 1-2 1-2 1-2 1-2 1-21-2 1-2 1-2 1-2 Group 2 F173 1-2 1-2 1-2 1-2 1-2 1-2 2 1-2 2 2 1-2 1-21-2 1-2 Cpd 2 F165 1 1 1-2 1-2 1-2 1-2 1-2 1-2 1-2 1-2 1-2 1-2 1-2 1-2300 mg/kg F177 1 1-2 1-2 1-2 1-2 1-2 1-2 1-2 1-2 1-2 1-2 1-2 1-2 1-2F187 1 1-2 1-2 1-2 1-2 1-2 2 1-2 1-2 1-2 1-2 1-2 1-2 2 F189 1 1 1-2 1-21-2 1-2 1-2 1-2 1-2 1-2 1-2 1-2 1-2 2

Two animals in the vehicle control group (F139 and F170) had a marginalnet loss in body weight from Day 1 to Day 14 of the study. One animal inthe drug group (F187) also showed a small decrease in body weight.

Study procedures, drugs and drug vehicles have varying affects on thehealth of study animals. In the current study, only 3 of 10 animalsshowed any degree of adverse impact and, on balance, this was equallydistributed between the control and drug groups. Thus, the currentdosing regimen and drug dose of 300 mg/kg was well tolerated by animals.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, documents,manuscripts and scientific literature cited herein are herebyincorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims. It should also be understood thatthe embodiments described herein are not mutually exclusive and thatfeatures from the various embodiments may be combined in whole or inpart in accordance with the invention.

We claim: 1-10. (canceled)
 11. A method of treating HCV infection in apatient, the method comprising administering to the patient in need ofsuch treatment an effective amount of a compound selected from the groupconsisting of compound 4, compound 6m, or compound 7:

or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer,solvate, or combination thereof.
 12. The method of claim 11, wherein thecompound is compound 4:

or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer,solvate, or combination thereof.
 13. The method of claim 11, wherein thecompound is compound 6m:


14. The method of claim 11, wherein the compound is compound 7:

or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer,solvate, or combination thereof.
 15. A method of treating HCV infectionin a patient, the method comprising administering to the patient in needof such treatment an effective amount of a compound of formula A1:

or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer,solvate, or combination thereof, wherein V, M, R¹⁰, and R¹¹ aredelineated for compounds 1-8 in Table 1: TABLE 1 Compound No. R X₁ R₃ R₄1

absent H H 2

O H H 3

absent H H 4

O H H 5

O C(O)Ph H 6

O H C(O)Ph 7

absent H H 8

O H H

and the compound is administered in combination with a second anti-HCVagent selected from a polymerase inhibitor and a protease inhibitor. 16.The method of claim 15, wherein the compound is compound 1:

or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer,solvate, or combination thereof.
 17. The method of any one of claims15-16, wherein the polymerase inhibitor or polymerase inhibitorcomprises ribavirin, an interferon, a pegylated interferon, levovirin,viramidine, thymosin-alpha-1, telaprevir, or a 2′-C-branchedribonucleoside.
 18. The method of claim 17, wherein the second anti-HCVagent is ribavirin.
 19. The method of claim 17, wherein the secondanti-HCV agent is an interferon.
 20. The method of claim 19, wherein theinterferon is interferon-α or interferon-β.
 21. The method of claim 17,wherein the second anti-HCV agent is a pegylated interferon.
 22. Themethod of claim 21, wherein the pegylated interferon is peginterferon-α.23. The method of claim 17, wherein the second anti-HCV agent istelaprevir.
 24. The method of claim 17, wherein the second anti-HCVagent is a 2′-C-branched ribonucleoside.
 25. The method of claim 24,wherein the 2′-C-branched ribonucleoside comprises 2′-C-methyl-cytidine,2′-C-methyl-uridine, 2′-C-methyl-adenosine, 2′-C-methyl-guanosine, or9-(2-C-methyl-β-D-ribofuranosyl)-2,6-diaminopurine.
 26. The method ofclaim 17, wherein the HCV infection is an HCV co-infection with a secondvirus.
 27. The method of claim 26, wherein the second virus is HBV, HIV,Influenza A, Influenza B, West Nile virus, Dengue virus, RSV, orrhinovirus.